Tagging with green fluorescent protein reveals a distinct subcellular distribution of L-type and non-L-type Ca2+ channels expressed in dysgenic myotubes.
Expression of cardiac L-type Ca2+ channels in dysgenic myotubes results in large Ca2+ currents and electrically evoked contractions resulting from Ca2+-entry dependent release of Ca2+ from the sarcoplasmic reticulum. By contrast, expression of either P/Q-type or N-type Ca2+ channels in dysgenic myotubes does not result in electrically evoked contractions despite producing comparably large Ca2+ currents. In this work we examined the possibility that this discrepancy is caused by the preferential distribution of expressed L-type Ca2+ channels in close apposition to sarcoplasmic reticulum Ca2+ release channels. We tagged the N termini of different alpha1 subunits (classes A, B, C, and S) with a modified green fluorescent protein (GFP) and expressed each of the fusion channels in dysgenic myotubes. Each GFP-tagged alpha1 subunit exhibited Ca2+ channel activity that was indistinguishable from its wild-type counterpart. In addition, expression of GFP-alpha1S and GFP-alpha1C in dysgenic myotubes restored skeletal- and cardiac-type excitation-contraction (EC) coupling, respectively, whereas expression of GFP-alpha1A and GFP-alpha1B failed to restore EC coupling of any type. Laser-scanning confocal microscopy revealed a distinct expression pattern for L-type compared with non-L-type channels. After injection of cDNA into a single nucleus, GFP-alpha1S and GFP-alpha1C were present in the plasmalemma as small punctate foci along much of the longitudinal extent of the myotube. In contrast, GFP-alpha1A and GFP-alpha1B were not concentrated into punctate foci and primarily were found adjacent to the injected nucleus. Thus, L-type channels possess a targeting signal that directs their longitudinal transport and insertion into punctate regions of myotubes that presumably represent functional sites of EC coupling.
Grabner
M
M
Department of Anatomy and Neurobiology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA.
Dirksen
R T
RT
Beam
K G
KG
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins; D015640:Ion Channel Gating; D008164:Luminescent Proteins; D051379:Mice; D018613:Microscopy, Confocal; D009119:Muscle Contraction; D009132:Muscles / Q000378:metabolism* / Q000648:ultrastructure; D009206:Myocardium / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D011993:Recombinant Fusion Proteins
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium